T cell activation involves the integration of several signals. In addition to antigen specific signaling through the T cell receptor (TCR) and costimulatory signaling through molecules such as CD28, signaling through additional molecules including the transmembrane receptor Notch are critical at many stages of T cell activation and development. To identify novel molecules that regulate T cell activation, we used a genetic approach to rescue the T cell activation defect in a Jurkat mutant cell line by retroviral expression of a leukocyte library. In our initial screen, we found that retroviral expression of NKAP, a protein whose function was poorly understood, complemented the defect in one Jurkat mutant cell line and restored T cell activation. We have demonstrated that NKAP is a novel negative regulator of Notch signaling. Overexpression of NKAP inhibits activation of the Notch transcriptional reporter CSL-luc, similar to dominant negative MAML. We have shown that NKAP associates with CIR, which is part of the Notch co- repressor complex. NKAP colocalizes with CIR in a punctate nuclear pattern, which is shared by many regulators of Notch activation including CSL and MAML. By using quantitative PCR, NKAP levels were found to be dynamically modulated by two orders of magnitude during T cell development, and its expression inversely correlated with the expression of a Notch target gene, Deltex, as would be expected for an antagonist of Notch activation. Through a combination of genetic and biochemical approaches, this proposal will focus on understanding the function of NKAP in Notch signaling, and its role at different stages of lymphocyte development and activation. Specific Aim 1: Understand the function of NKAP during T cell development and activation Specific Aim 2: Identification of genes directly regulated by NKAP Specific Aim 3: Function of NKAP within the corepressor complex to Public Health: Notch signaling is critical to the generation and activation of T cells, which regulate immune responses, whether beneficial, as in the elimination of pathogens, or detrimental, as occurs during autoimmunity. This proposal will focus on understanding how NKAP, a negative regulator of Notch signaling, affects lymphocyte development, activation and effector function. [unreadable] [unreadable] [unreadable]